The goal of the studies described in this application is to use isolated bovine adrenal chromaffin cells to understand the role of the multiple-site phosphorylation of tyrosine hydroxylase (TH) in the physiological regulation of catecholamine biosynthesis. These goals will be pursued in three phases. First, TH from control and acetylcholine treated cells will be purified and the sites phosphorylated on TH separated using proteolytic digestion and HPLC. To verify the uniqueness of each phosphorylation site, amino acid sequence determinations will be carried out. To predict the position of the phosphorylation sites within the TH molecule, the amino acid sequences flanking the phosphorylation sites will be compared with the TH sequences derived from rat cDNA (Grima et al., Proc.Natl.Acad.Sci. 82:617, 1985). To complete the analysis of the in situ phosphorylation of TH, the phosphate content of each unique phosphorylation site in basal and acetylcholine stimulated cells will be determined. In the second phase of the project, the HPLC retention characteristics of the unique phosphorylation sites will be used as a guide in determining the identify of the protein kinase activities responsible for the in situ phosphorylation of TH. Pharmacological experiments using drugs which stimulate or inhibit known protein kinases will be conducted to verify the role of each candidate kinase in TH phosphorylation and activation in situ. In the third phase of the project the influence of combinations of cholinergic and peptidergic agonists on the phosphorylation and activation of TH in the isolated cells will he examined to rest the hypothesis that the phosphorylation of TH on multiple sites is part of a mechanism to integrate the influence of multiple inputs to adrenergic cells.